An experiment created for the purposes of harvesting ADAM, the Little Sisters largely represent the morality aspect of the game. Upon destroying a Sister's guardian Big Daddy, the player can choose to harvest the Sister for a lot of ADAM (and hence Plasmids, Tonics, and everything else the Gatherer's Garden offers), or rescue them for less (but with rewards from Tenenbaum for every three rescued).

As Little Sisters are vulnerable to attack from Splicers and attract them with the ADAM they create, Suchong created the Big Daddies to protect them. Little Sisters are attracted to Big Daddies by the sight of them, the sound of them, and the smell of a special pheremone created for Big Daddies.

Little Sisters were created by embedding ADAM slugs into their stomachs. They were then mentally conditioned by Dr. Suchong to see ADAM filled corpses as "angels," where they would extract the ADAM from throughout the city.

Little Sisters travel throughout the world of Rapture via special vents.